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Application of SYBR Green real-time PCR assay for the specific detection of Salmonella spp.

Salmonella spp. 특이적인 검출을 위한 SYBR Green real-time PCR 기법 적용

  • Shin, Seung Won (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Cha, Seung Bin (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Lee, Won-Jung (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Shin, Min-Kyoung (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Jung, Myunghwan (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Yoo, Anna (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University) ;
  • Jung, Byeng Yeal (Bacterial Disease Division, Department of Animal and Plant Health Research, Animal, Plant and Fisheries Quarantine and Inspection Agency) ;
  • Yoo, Han Sang (Department of Infectious Diseases, College of Veterinary Medicine and Brain Korea 21 Program for Veterinary Science, Seoul National University)
  • 신승원 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 차승빈 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 이원정 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 신민경 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 정명환 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 유안나 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단) ;
  • 정병열 (농림수산검역검사본부 동식물위생연구부 세균질병과) ;
  • 유한상 (서울대학교 수의과대학 및 BK21 수의과학인력양성 사업단)
  • Received : 2012.08.09
  • Accepted : 2012.12.14
  • Published : 2013.03.31

Abstract

The aim of this study was to applicate and evaluate a SYBR Green real-time PCR for the specific detection of Salmonella spp. Specificity of the PCR method was confirmed with 48 Salmonella spp. and 5 non-Salmonella strains using invA gene primer. The average threshold cycle ($C_T$) of Salmonella spp. was $11.83{\pm}0.78$ while non-Salmonella spp. was $30.86{\pm}1.19$. Correlation coefficients of standard curves constructed using $C_T$ versus copy number of Salmonella Enteritidis ATCC 13076 showed good linearity ($R^2=0.993$; slope = 3.563). Minimum level of detection with the method was > $10^2$ colony forming units (CFU)/mL. These results suggested that the SYBR Green real-time PCR might be applicable for the specific detection of Salmonella spp. isolates.

Acknowledgement

Supported by : 농림수산식품기술기획평가원

References

  1. Perry JD, Freydiere AM. The application of chromogenic media in clinical microbiology. J Appl Microbiol 2007, 103, 2046-2055. https://doi.org/10.1111/j.1365-2672.2007.03442.x
  2. Rahn K, De Grandis SA, Clarke RC, McEwen SA, Galan JE, Ginocchio C, Curtiss R 3rd, Gyles CL. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol Cell Probes 1992, 6, 271-279. https://doi.org/10.1016/0890-8508(92)90002-F
  3. Rodriguez-Lazaro D, Hernandez M, Esteve T, Hoorfar J, Pla M. A rapid and direct real time PCR-based method for identification of Salmonella spp. J Microbiol Methods 2003, 54, 381-390. https://doi.org/10.1016/S0167-7012(03)00071-X
  4. Tirado C, Schmidt K. WHO surveillance programme for control of foodborne infections and intoxications: preliminary results and trends across greater Europe J Infect 2001, 43, 80-84. https://doi.org/10.1016/S0163-4453(01)90861-8
  5. Waltman WD, Mallinson ET. Isolation of Salmonella from poultry tissue and environmental samples: a nationwide survey. Avian Dis 1995, 39, 45-54. https://doi.org/10.2307/1591981
  6. Daum LT, Barnes WJ, McAvin JC, Neidert MS, Cooper LA, Huff WB, Gaul L, Riggins WS, Morris S, Salmen A, Lohman KL. Real-time PCR detection of Salmonella in suspect foods from a gastroenteritis outbreak in Kerr County, Texas. J Clin Microbiol 2002, 40, 3050-3052. https://doi.org/10.1128/JCM.40.8.3050-3052.2002
  7. De Medici D, Croci L, Delibato E, Di Pasquale S, Filetici E, Toti T. Evaluation of DNA extraction methods for use in combination with SYBR Green I real-time PCR to detect Salmonella enterica serotype Enteritidis in poultry. Appl Environ Microbiol 2003, 69, 3456-3461. https://doi.org/10.1128/AEM.69.6.3456-3461.2003
  8. Eijkelkamp JM, Aarts HJM, van der Fels-Klerx HJ. Suitability of rapid detection methods for Salmonella in poultry slaughterhouses. Food Anal Methods 2009, 2, 1-13. https://doi.org/10.1007/s12161-008-9040-5
  9. Eriksson E, Aspan A. Comparison of culture, ELISA and PCR techniques for salmonella detection in faecal samples for cattle, pig and poultry. BMC Vet Res 2007, 3, 21. https://doi.org/10.1186/1746-6148-3-21
  10. Foley B, McKeown P, de Lappe N, Cormican M. Salmonellosis in Ireland, 2006. EPI Insight 2007, 8, 2-3.
  11. Fukushima H, Tsunomori Y, Seki R. Duplex real-time SYBR Green PCR assays for detection of 17 Species of foodor waterborne pathogens in stools. J Clin Microbiol 2003, 41, 5134-5146. https://doi.org/10.1128/JCM.41.11.5134-5146.2003
  12. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996, 6, 986-994. https://doi.org/10.1101/gr.6.10.986
  13. Hoorfar J, Ahrens P, Radstrom P. Automated 5' nuclease PCR assay for identification of Salmonella enterica. J Clin Microbiol 2000, 38, 3429-3435.
  14. Jothikumar N, Griffiths MW. Rapid detection of Escherichia coli O157:H7 with multiplex real-time PCR assays. Appl Environ Microbiol 2002, 68, 3169-3171. https://doi.org/10.1128/AEM.68.6.3169-3171.2002
  15. Jothikumar N, Wang X, Griffiths MW. Real-time multiplex SYBR Green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes. J Food Prot 2003, 66, 2141-2145. https://doi.org/10.4315/0362-028X-66.11.2141
  16. Knutsson R, Lofstrom C, Grage H, Hoorfar J, Radstrom P. Modeling of 5' nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica. J Clin Microbiol 2002, 40, 52-60. https://doi.org/10.1128/JCM.40.1.52-60.2002
  17. Liming SH, Bhagwat AA. Application of a molecular beacon-real-time PCR technology to detect Salmonella species contaminating fruits and vegetables. Int J Food Microbiol 2004, 95, 177-187. https://doi.org/10.1016/j.ijfoodmicro.2004.02.013
  18. Nam HM, Srinivasan V, Gillespie BE, Murinda SE, Oliver SP. Application of SYBR Green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples. Int J Food Microbiol 2005, 102, 161-171. https://doi.org/10.1016/j.ijfoodmicro.2004.12.020
  19. Newton-Clarke M. Principles of prevention and control of salmonellosis. Equine Vet Educ 1995, 7, 67-69. https://doi.org/10.1111/j.2042-3292.1995.tb01192.x
  20. Nogva HK, Lillehaug D. Detection and quantification of Salmonella in pure cultures using 5'-nuclease polymerase chain reaction. Int J Food Microbiol 1999, 51, 191-196. https://doi.org/10.1016/S0168-1605(99)00122-1
  21. Oosterom J. Epidemiological studies and proposed preventive measures in the fight against human salmonellosis. Int J Food Microbiol 1991, 12, 41-51. https://doi.org/10.1016/0168-1605(91)90046-R
  22. Aarts HJM, Joosten RG, Henkens MHC, Stegeman H, van Hoek AHAM. Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains. J Microbiol Methods 2001, 47, 209-217. https://doi.org/10.1016/S0167-7012(01)00305-0
  23. Bhagwat AA. Rapid detection of Salmonella from vegetable rinse-water using real-time PCR. Food Microbiol 2004, 21, 73-78. https://doi.org/10.1016/S0740-0020(03)00020-0
  24. Carli KT, Unal CB, Caner V, Eyigor A. Detection of Salmonellae in chicken feces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis. J Clin Microbiol 2001, 39, 1871-1876. https://doi.org/10.1128/JCM.39.5.1871-1876.2001
  25. Chen S, Yee A, Griffiths M, Larkin C, Yamashiro CT, Behari R, Paszko-Kolva C, Rahn K, De Grandis SA. The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int J Food Microbiol 1997, 35, 239-250. https://doi.org/10.1016/S0168-1605(97)01241-5
  26. Chen W, Martinez G, Mulchandani A. Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella. Anal Biochem 2000, 280, 166-172. https://doi.org/10.1006/abio.2000.4518
  27. Chiu TH, Chen TR, Hwang WZ, Tsen HY. Sequencing of an internal transcribed spacer region of 16S-23S rRNA gene and designing of PCR primers for the detection of Salmonella spp. in food. Int J Food Microbiol 2005, 97, 259-265. https://doi.org/10.1016/j.ijfoodmicro.2004.04.005
  28. Cogan TA, Humphrey TJ. The rise and fall of Salmonella Enteritidis in the UK. J Appl Microbiol 2003, 94 (Suppl), 114S-119S. https://doi.org/10.1046/j.1365-2672.94.s1.13.x
  29. Crump JA, Luby SP, Mintz ED. The global burden of typhoid fever. Bull World Health Organ 2004, 82, 346-353.